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biotechnology

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is the use, and especially the alteration, of organisms, cells, or biological molecules to produce food, drugs, or other goods Traditional applications of biotechnology include the use of yeast to make bread, beer, and wine, and the selective breeding of animals

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More modern uses modify the genes directly through genetic engineering.

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A key tool of genetic engineering is recombinant DNA, which is DNA that has been altered to contain genes or portions of genes from different organisms Plants and animals that express DNA that has been modified or derived from other species are transgenic or genetically modified organisms (GMOs)

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Recombinant DNA

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DNA that has been altered to contain genes or portions of genes from different organisms.

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What recombines DNA?

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Sexual reproduction

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During meiosis 1, what does the homologous chromosomes do?

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They exchange DNA, Therefore, every egg and every sperm contain recombinant DNA that is derived from the two parents

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Transformation may combine DNA from ___ ____ ___

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different bacterial speciesl.

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What happens in transformation?

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bacteria pick up pieces of DNA from the environment. This DNA could be part of a chromosome from another bacterium or tiny circular DNA molecules called plasmids Passing plasmids from bacteria to yeast may also occur, a process that moves genes from prokaryotes to eukaryotes

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What are plasmids?

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Plasmids are small pieces of DNA, not included in the chromosomes, that may be present in bacteria in multiple copies Plasmids carry genes that help the bacteria survive in novel environments, such as where a new energy source is available or where antibiotics are present

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Viruses

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are little more than genetic material encased in a protein coat, and can only reproduce inside cells

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Viral reproduction (what are the 5 steps)

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-A virus first attaches to specific molecules on the surface of a suitable host cell -The virus enters the cytoplasm of the host cell -The virus releases its genetic material The host replicates the viral genetic material and synthesizes viral proteins -The replicated genes and viral proteins assemble inside the cell -New viruses are formed, which are released and may infect new cells

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The life cycle of a typical virus

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1. A virus attaches to a susceptible host cell 2.The virus enters the host cell 3.The virus releases its DNA into the host cell; some viral DNA (red) may be incorporated into the host cell's DNA (blue) 4.Viral genes encode the synthesis of viral proteins and viral gene replication; some host cell DNA may attach to the replicated viral DNA (red/blue combination) 5.New viruses assemble; some host cell DNA is carried by recombinant viruses 6.The host cell bursts open, releasing newly assembled viruses; if recombinant viruses infect a second cell, they may transfer genes from the first cell to the second cell

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Can viruses transfer DNA between species?

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Yes

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In some instances, viral DNA is inserted into one of the host cell's chromosomes . When new viruses are produced, what happens?

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some of the host cell's genes may be attached to the viral DNA

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If such a virus infects another host cell, its DNA and pieces of the previous host cell's DNA will ______

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also be inserted

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Why do most viruses infect specific host species?

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so that they move host DNA between different individuals of a single, or fairly closely related, species Some viruses can infect multiple species

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what can Gene transfer do?

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between viruses that infect multiple species can produce extremely lethal recombined viruses, as occurred when bird flu and human flu viruses recombined and caused global epidemics and hundreds of thousands of deaths in 1957 and 1968

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What amplifies DNA?

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The polymerase chain reaction

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What did Kary mullus of the Cetus corporation do?

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he developed the polymerase chain reaction (PCR) produces virtually unlimited copies of a very small DNA sample

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What can PCR (polymerase chain reaction) do?

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can target a desired sequence of DNA in a sample and amplify it to a quantity that can be selected out and used

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What is PCR used for?

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is used by forensic scientists to identify victims and criminals, and is used extensively in biotechnology and biomedicine

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What does PCR require?

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small pieces of DNA (called primers) that are complementary to the gene sequences targeted for copying

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What are primers?

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small pieces of DNA . One primer is complementary to one strand of the double helix and the other primer is complementary to the other strand One of the two primers pairs at one end of the targeted area; the other primer pairs to the other end of the targeted area In actual DNA replication, primers are necessary to give DNA polymerase a place from which to build

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What is a PCR \"run\" ?

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Basically a DNA replication in a tiny test tube. -Template DNA, primer, nucleotides, and DNA polymerase are all in the reaction mix The DNA polymerase (Taq polymerase) is a special enzyme that can function at the relatively high temperatures required for PCR

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a PCR cycle involves 4 steps, what are they?

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-The test tube is heated to 90-95ºC to cause the double-stranded template DNA to separate into single strands -The temperature is lowered to 50ºC to allow the two primers to form complementary base pairs with the two original DNA strands -The temperature is raised to 70-72ºC so that DNA polymerase can use the free nucleotides to make copies of the DNA segment bounded by the primers -This cycle is repeated, usually 30 to 40 times, until the reactants are used up

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PCR Copies a specific DNA sequence

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1. Heating separates DNA strands (80C or 194F) 2. Cooling allows primers and DNA polymerase to bind (60C or 122 F) 3. Now DNA strands are synthesized (158F or 70C)

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Differences in short tandem repeats can identify individuals by their DNA

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Forensic scientists have found that small, repeating segments of DNA, called short tandem repeats (STRs), can be used with astonishing accuracy to identify people Each STR is short (consisting of 2 to 5 nucleotides), repeated (as many as 5 to 50 times), and tandem (having all the repetitions right alongside one another) STRs are repeated sequences of DNA within the chromosomes that do not code for proteins

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STRs vary greatly between ____. give an example.

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human individuals, i.e genetic fingerprints.

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Different people may have different alleles of the STRs In the case of an STR, each allele simply has a different number of repeats of the same few nucleotides

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The U.S. Department of Justice established a standard set of 13 STRs, each four nucleotides long, to identify individuals by DNA samples There may be as few as 5 to as many as 38 repeats of these STRs

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Mixtures of DNA fragments can be separated in the basis

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of size

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Gel elctrophoresisis

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A technique used to spread out DNA fragments of varying lengths in mixture.

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there are 4 steps in gel electrophoresis, what are they?

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-DNA mixtures are placed into wells at one end of a slab of agarose gel, and an electric current is introduced to the gel -Because of its phosphates, the negatively charged DNA moves toward the positive electrode, with smaller fragments moving through the gel meshwork more quickly than larger ones The mixture is thus separated into bands of DNA along the gel, according to how many nucleotides long each DNA fragment is -When the smallest fragments have migrated to the end of the gel, the fragments are separated into single strands and a different current drives the separated bands of DNA down onto a nylon sheet -The nylon paper with the DNA bound to it is bathed in a solution of labeled DNA probes that are complementary to specific DNA segments of interest in the original DNA sample The targeted DNA fragments base-pair with the probe, which reveals their location on the nylon paper

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4 steps in gel electrophoresis and labeling with DNA probes separates and identifies segments of DNA

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- DNA samples are pipetted into wells (shallow slots) in the gel. Electrical current is sent through the gel (negative at the end with the wells and positive at the opposite end). - Electrical current moves the DNA segments through the gel. Smaller pieces of DNA move farther toward the positive electrode - The gel is placed on special nylon \"paper.\" Electrical current drives the DNA out of the gel onto the nylon. -The nylon paper with the DNA bound to it is bathed in a solution of labeled DNA probes (red) that are complementary to specific DNA segments in the original DNA sample. -Complementary DNA segments are labeled by the probes (red bands).

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What are DNA probes used for?

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used to label specific nucleotide sequence

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What are DNA probes?

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short, single-stranded DNA fragments used to identify DNA in a gel pattern The probe sequence is complementary to a DNA fragment somewhere in the gel pattern, so it is able to differentiate a particular stretch of DNA (such as a specific STR) from all the other DNA in the sample Probes are labeled with radioactivity or a colored dye and mark the location of a specific gene sequence when they base-pair with it

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Unrelated people almost never have identical DNA profiles

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Know this

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DNA samples run on STR gels produce a pattern, called what?

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a DNA profile.

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How are the positions of the band on gel determined?

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by the numbers of repeats in each STR allele

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If the same STRs are analyzed, then a particular DNA sample will produce what?

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the same profile everytime.

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What is the main goal of agriculture?

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to grow as much food as possible as cheaply with minimal loss from pest

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How do you clone a gene?

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-The desired gene is first isolated from the organism containing it The gene may alternatively be synthesized in the laboratory -The gene is next inserted into a plasmid, which replicates itself autonomously in bacteria in huge numbers; the copied gene is then transferred to a plant or animal

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What do biotechnologists use to infect plant cells?

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Ti plasmid from the bacterium Agrobacterium tumefaciens

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What is a Ti?

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Ti stands for tumor-inducing The tumor-inducing genes are removed when the plasmid is inserted The Ti plasmid, unlike most plasmids, inserts itself into the host's chromosomes, so that when plant cells are infected, they are more likely to pass the recombinant genes to future generations of cells

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Restriction enzymes cut DNA at specific nucleotide sequences

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...

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How are genes inserted into plasmids?

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through the action of restriction enzymes isolated from bacteria.

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Restriction enzymes

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nucleases that cut DNA at specific nucleiotide sequences.A DNA sequence (e.g., a gene) can be removed from a chromosome using these special enzymes -Some restriction enzymes cut straight across the double helix Some restriction enzymes make a staggered cut, snipping the DNA in a different location on each of the two strands The staggered cut leaves a short section of single-stranded DNA at each end These unpaired sections of single-stranded DNA are called sticky ends Enzymes that create staggered cuts in DNA with \"sticky ends\" are the most useful in gene cloning because they can stick to other single-stranded pieces of DNA with complementary bases (i.e., DNA cut with the same restriction enzyme)

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Cutting two pieces of DNA with the same restriction enzyme allows what?

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the pieces to be joined Sticky ends allow for splicing a DNA fragment with another complementary fragment The Bt gene can be cut out of the Bacillus chromosome with the same enzyme used to cut open the Ti plasmid Therefore, when mixed Bt gene fragment ends can base-pair with sticky ends of the opened plasmid, adding the gene to the plasmid circle DNA ligase enzyme is then used to bond the gene permanently into the plasmid

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DNA ligase

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Enzme is used to bond the gene permanently into the plasmid.

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Plasmid-transformed bacteria insert the Bt gene into a plant

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Agrobacterium tumefaciensis is transformed with the recombinant Ti plasmid containing the Btgene Agrobacterium infects plant cells and inserts its small Ti plasmid into a plant chromosome in the nucleus Hormones induce the transgenic plant cells containing the Bt gene to differentiate into full plants

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Alternatively, a \"gene gun\" can be used to insert recombinant DNA into plant cells

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The desired DNA, often in a plasmid, is coated on tiny metallic particles The particles are fired into a lump of plant cells Even though very few plant cells actually incorporate the new gene into a chromosome, only a few have to in order to generate viable plants Despite its \"hit or miss\" nature, the method is so straightforward, it is being increasingly used to produce genetically modified plants

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Using Plasmids to Insert a Bacterial Gene into a Plant Chromosome

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1.The DNA containing the Bt gene and the Ti plasmid are cut with the same restriction enzyme. 2.Bt genes and Ti plasmids, both with the same complementary sticky ends, are mixed together; DNA ligase bonds the Bt genes into the plasmids. 3.Bacteria are transformed with the recombinant plasmids. 4. Transgenic bacteria enter the plant cells, and Bt genes are inserted into the chromosomes of the plant cells.

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Genetically modified plants may be used to produce medicines Medically useful genes can be inserted into plants

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Plants have been engineered to produce harmless hepatitis B virus and E. coli proteins, stimulating an immune response when eaten Plants could be engineered to produce human antibodies, conferring passive immunity to microbial infection merely by eating the plant

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Goats have been engineered to carry genes for spider silk, which appears in their milk and produces a meshwork stronger than steel or Kevlar

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Sheep can be engineered to produce medically important proteins in their milk that are useful in treating cystic fibrosis or blood clotting factors

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Findings from the Human Genome Project:

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There are 20,000 to 25,000 human genes Genes make up only 2% of the total DNA New genes, including many disease-associated genes, were discovered, but it's not really known what most of DNA does

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Why was this project undertaken?

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Knowing the DNA sequence of a novel gene may allow biologists to predict the function of the protein the gene encodes Knowing the sequence of a gene allows predictions about what the amino acid sequence of the encoded protein will be The amino acid sequence can then be compared with proteins whose functions are already known, and if matches are found, inferences can be made about the new gene's function Knowing the nucleotide sequences of human genes will aid in the treatment of many diseases

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Even though most human DNA is the same for everyone on the planet, examining the genomes of many individuals uncovers unique sets of alleles in individuals These may correlate with their observed medical conditions, predispositions to disease, and differential responses to treatment Knowledge of these individual differences is already allowing treatment protocols to be refined to take the differences into account

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Comparison of our genome with those of other species will clarify the genetic differences that help to make us human

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DNA technology can be used to diagnose inherited disorders Two methods are commonly used to find out if a person carries a normal allele or a malfunctioning allele what are they?

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Restriction enzymes may cut different alleles of a gene at different locations, yielding distinctive fragments characteristic of one allele or the other Different alleles bind to different DNA probes If homologous chromosomes have different alleles at several loci, the two chromosomes may produce different numbers of fragments, as well as fragments of different length, when cut by restriction enzymes These differences, called restriction fragment length polymorphisms (RFLPs), can be detected, and the patterns observed can be used to diagnose the presence or absence of various alleles

Biology 111 chapter 13

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